ABSTRACT Glaucoma is the most prevalent optic neuropathy where a progressive degeneration of retinal ganglion cells (RGCs) leads to vision loss. Our long-term goal is to help prevent the degeneration of glaucomatous RGCs by characterizing pluripotent stem cells as a renewable source of RGCs for autologous ex vivo cell therapy. The objective of this renewal application is to address the next question relevant to the potential clinical application of human pluripotent cell-derived RGCs: whether or not these cells can elaborate guidable axons that can navigate out of the host retina and seek bonafide targets, essential for reversing vision loss. To our knowledge this question, essential for practical ex-vivo stem cell approach to glaucomatous degeneration, remains unanswered. The central hypothesis of the proposed study is that human induced pluripotent stem cells derived RGCs (hiPSC-RGCs) elaborate guidable axons, regulated by the mTOR pathway, an intrinsic regulator axonogenesis and regeneration. Our reasoning is based on our observations that hiPSC-RGCs are (1) stable, functional, and safe (2) express guidance receptors and respond to both proximal (intra- retinal) and distal (extra-retinal) guidance cues, and (3) have active mTOR pathway, regulating development and neuritogenesis. Our rationale is that the ability of hiPSC-RGCs to recapitulate the mechanism of axon growth and guidance will posit them as a viable reagent to functionally replace degenerated RGCs in glaucoma. The following specific aims are proposed to test the hypothesis: Aim 1: To determine the competence of hiPSC-RGCs for axon guidance and target specificity, Aim 2: To determine the competence of hiPSC-RGCs for mTOR-dependent axonogenesis and regeneration in vitro, and Aim 3: To determine mTOR-dependent hiPSC-RGC axonogenesis in neonatal and adult retina. The potential of hiPSC-RGCs for axonogenesis and axon guidance will be examined in co-culture paradigm using the microfluidic system in controlled conditions. Immunocytochemical analysis of known pathways and transcriptional profiling would identify candidate regulatory factors. The regenerative ability of hiPSC-RGCs in the context of mTOR pathway will be examined in a microfluidic model of the axotomy model, established in our lab. Transcription profile at pre-axotomy, axotomy, and post-axotomy stages would identify regenerative gene regulatory network. Finally, regenerative capacity of hiPSC-RGCs and the influence of the mTOR pathway will be examined in vivo in neonatal retina, where environment is conducive for axon growth and in a degenerative adult environment in animal model of glaucoma. Our research proposal is innovative because it will determine whether the de novo generated neurons can functionally replace those that make long distance connections such as RGCs and bridge a gap in our knowledge about human RGC development and axon path finding, a barrier to optic nerve regeneration. The emerging information will be significant because it will not only address each of the most significant barriers that currently make the ex-vivo stem cell therapy approach impractical but also lead to the development of a robust model system for testing normal/pathological mechanisms of RGC development and for screening drugs and genes for additional new therapeutic approaches for glaucomatous retinal degeneration.